DNA Marker Analysis Research Articles

Prenatal diagnosis and molecular cytogenetic characterization of a de novo interstitial duplication of 11q (11q22.3→q23.3) associated with abnormal maternal serum biochemistry

ObjectiveTo present prenatal diagnosis and molecular cytogenetic characterization of a de novo interstitial duplication of 11q (11q22.3→q23.3) in a pregnancy associated with abnormal maternal serum biochemistry. Case ReportA 33-year-old woman underwent amniocentesis in the second trimester because of abnormal maternal serum biochemistry. Her husband was 33 years old. At 17 weeks of gestation, the levels of α-fetoprotein (AFP), unconjugated estriol (uE3), total β-human chorionic gonadotropin (β-hCG), and inhibin A were 0.65 multiples of median (MoM), 0.61 MoM, 0.32 MoM, and 0.55 MoM, respectively, consistent with a positive trisomy 18 risk of 1/128. Results of amniocentesis revealed a small de novo interstitial duplication of 11q encompassing 11q23. An array comparative genomic hybridization analysis detected a 9.04-Mb duplication at chromosome 11q22.3-q23.3. A polymorphic DNA marker analysis was carried out, which determined a paternal origin of the duplication. Results of fluorescence in situ hybridization analysis showed a direct duplication of interstitial 11q. The karyotype was 46,XX,dup(11)(q22.3q23.3). Level II ultrasound was unremarkable. The parents opted to continue the pregnancy. A 2792-g female baby was delivered at 38 weeks of gestation. When examined at 10 months of age, the neonate was small for age and was abnormal in psychomotor development with apparent facial dysmorphisms, and small hands and feet. ConclusionLow levels of AFP, uE3, β-hCG, and inhibin A in the second trimester maternal serum biochemistry may be a distinctive prenatal feature in pregnancy with fetal chromosome 11q duplication.

Microsatellite DNA Marker Analysis in Brown Mussels, Perna perna (Linnaeus, 1758) from the Coastal Waters of Oman

Thirteen highly polymorphic microsatellite primer pairs developed for Perna viridis from the gene bank were tested on 3 populations of Perna perna samples with the aim to adapt a fast, reliable method for preliminary screening and to genetically characterize the wild populations of P. perna in the Oman waters. The samples were collected from three different locations viz Ras Al-Had, Ras Madrakah and Mirbat. All primers showed high level of polymorphism for all populations. The mean observed heterozygosity was lower than the expected heterozygosity across the three populations which means there was a probability of inbreeding occurred in the populations. Both the chi-square (χ2) and likelihood (G2) ratio tests detected significant differences (p<0.05), which showed deviations from Hardy-Weinberg equilibrium. Cluster analysis revealed a close genetic relationship of P. perna between all populations and they were clustered according to their geographical origins into two major groups which include one cluster that grouped the Mirbat and the Ras Madrakah populations together while the other cluster showed the Ras Al Had domain. The highest genetic distance (1.2666) was observed between the Mirbat and the Ras Al Had populations while the lowest genetic distance (0.4746) was recorded between the Mirbat and Ras Madrakah populations. This study demonstrated that microsatellite markers with thirteen P. viridis primer pairs tested can be applied to genetically characterize the brown mussel populations in Oman waters.

POPULATION GENETIC STRUCTURE OF AN ENDANGERED KALIBAUS, Labeo calbasu (HAMILTON, 1822) REVEALED BY MICROSATELLITE DNA MARKERS

The population genetic structure of Labeo calbasu collected from four wild and a hatchery population was studied using microsatellite DNA marker analysis. Five heterologous microsatellite markers (Lr10, Lr21, Lr24, Lr26 and CcatG1) developed from rohu and catla were analyzed to test the genetic variability of L. calbasu stocks. The number of alleles observed in the loci ranged from 2-10. The loci were found to be polymorphic (<P95) in all the populations. The average numbers of possessed alleles were higher by the four wild stocks than the hatchery stock. The average number of allele was the highest in the Jamuna population (5.8) and the least in the Hatchery population (4.8). The observed average heterozygosity (Ho) in the Jamuna population (0.776) was the highest followed by the Halda (0.667), the Haor (0.661) and the Padma (0.642) populations. Except loci Lr10 and Lr24 in the Halda and locus Lr10 in the Padma and Hatchery populations, significant deviations from Hardy-Weinberg Equilibrium (HWE) were detected in all cases. The FST values and the Nm values indicated high level of differentiation and a low level of gene flow between the populations. The largest genetic distance value (D = 0.543) was measured between the Jamuna and the Hatchery populations while the least value (D =0.124) was observed between the Padma and the Halda populations. The estimated genetic population structure and potential applications of microsatellite markers may assist the proper management of L. calbasu populations in the wild.

Identification of disomic alien chromosome addition lines and a substitution line from monosomic alien addition lines of Chinese kale – flowering Chinese cabbage

SummaryGeneration of disomic alien chromosome addition lines and alien chromosome substitution lines is important for genetic research and chromosome engineering. After determining the transmission rates for alien chromosomes, we created five Chinese kale–flowering Chinese cabbage disomic alien addition lines and one substitution line. Transmission rates ranged from 11.46 – 38.18% via the female gametes, and from 4.88 – 12.90% through the male gametes. The disomic addition lines (CC+2A2, CC+2A3, CC+2A6, CC+2A8, and CC+2A9) were produced from selfed progenies of their corresponding monosomic addition lines. The substitution line C1/A9 was also obtained from the selfed progeny of monosomic line CC+A9, following cytological identification and simple sequence repeat DNA marker analysis.

Elucidation of Genetic Relationships in the Genus Cajanus Using Random Amplified Polymorphic DNA Marker Analysis

In this present investigation, a total of 27 RAPD primers were used to elucidate the genetic relationships between cultivated Cajanus cajan cultivars and 10 allied species of primary, secondary and tertiary gene pool, including C. cajanifolius, C. scarabaeoides, C. platycarpus, C. albicans, C. volubilis, C. sericeus, C. acutifolius, C. lineatus, C. lanceolatus and C. reticulates. All primers showed polymorphism at species level and produced 215 unequivocal polymorphic bands, with an average of 7.96 bands per primer. These polymorphic primers exhibited variation with regard to average band informativeness, resolving power, and showed high polymorphism information content value. No single primer was able to distinguish between all the two cultivars of C. cajan and ten allied species of Cajanus, but several species specific amplified fragments were observed. The pair wise Jaccard’s similarity coefficient values revealed high level of inter-specific genetic variation in the genus Cajanus. Cluster analysis exhibited the grouping of two C. cajan accessions with C. cajanifolius in one cluster, while except C. platycarpus, all the nine wild Cajanus species belonging to the secondary and tertiary gene pool form another cluster. The present analysis more or less agreed with the sectional classification of the genus Cajanus, and it has been hypothesized that cultivated pigeonpea has evolved through multi-genomic interaction through C. cajanifolius, and it has experienced minor genomic reorganization during its divergence. Again, identification of species specific amplification pattern substantiated the utility of RAPD markers on selection of suitable species to transfer the desirable trait into cultivated C. cajan, through marker aided breeding for its genetic augmentation, and also for the effective management of genetic resources of C. cajan.

Mitochondrial DNA and microsatellite markers evidence a different pattern of hybridization in red-legged partridge (Alectoris rufa) populations from NW Italy

Genetic introgression with exotic genomes represents a major conservation concern for the red-legged partridge (Alectoris rufa, Phasianidae). In particular, massive releases of chukar partridges (Alectoris chukar) and/or red-legged × chukar partridge hybrids for hunting purposes have cast serious doubts on the Italian present-day occurrence of wild A. rufa populations not affected by introgressive hybridization. This study investigates the genetic structure of red-legged partridges populations in two ecologically different areas in Northern Italy. Analysis of maternal mitochondrial DNA and biparental microsatellite markers excluded the presence of hybridization in a typical agricultural habitat where hunting and release of reared birds are strictly banned. By contrast, signs of chukar introgression were detected in a perifluvial habitat unusual for the red-legged partridge in Italy. The present study documents the first red-legged partridge population with no genetic evidences of recent chukar introgression presently living in Italy. We recommend that urgent conservation actions are taken to preserve the genetic integrity of this population from the risk of hybridization with farm-reared birds and to support its long-term conservation.

Mosaic trisomy 2 at amniocentesis: Prenatal diagnosis and molecular genetic analysis

ObjectiveThis study aims at presenting prenatal diagnosis of mosaic trisomy 2 and reviewing the literature. Materials, Methods, and ResultsA 32-year-old woman underwent amniocentesis at 21 weeks of gestation because of abnormal maternal serum biochemistry. Amniocentesis revealed a karyotype of 47,XY,+2[1]/46,XY[21] in in situ cultures. The single colony with trisomy 2 had two metaphase cells, and both had the karyotype of 47,XY,+2. Repeated amniocentesis was performed at 23 weeks of gestation. Interphase fluorescence in situ hybridization (FISH) analysis on uncultured amniocytes using a 2q11.1-specific probe RP11-468G5 (spectrum green) showed three green signals in 11 of 47 uncultured amniocytes, indicating 23.4% mosaicism for trisomy 2. The cultured amniocytes had a karyotype of 46,XY[20 colonies]. Polymorphic DNA marker analysis excluded uniparental disomy 2. The woman underwent the third amniocentesis at 25 weeks of gestation. Interphase FISH analysis on uncultured amniocytes revealed 9.4% (5/53 cells) mosaicism for trisomy 2. The cultured amniocytes had a karyotype of 46,XY[30 colonies]. Prenatal ultrasound was normal. The parents decided to continue the pregnancy to term, and a 3316-g baby was delivered with no phenotypic abnormalities. Cord blood had a karyotype of 46,XY[40 cells]. Interphase FISH analysis on uncultured urinary cells revealed 8.2% (4/49 cells) mosaicism for trisomy 2. The neonate was normal in growth and psychomotor development at 6 months of age. ConclusionPrenatal diagnosis of a single colony with two or more cells with trisomy 2 at amniocentesis should alert a clinically significant aneuploidy, and interphase FISH on uncultured amniocytes is useful for rapid confirmation of low-level trisomy 2 mosaicism at amniocentesis. The abnormal cell line of trisomy 2 may disappear after long-term amniocyte cultures.

Prenatal diagnosis of mosaic tetrasomy 18p

ObjectiveTo present prenatal diagnosis and molecular cytogenetic characterization of a small supernumerary marker chromosome derived from isochromosome 18p, by interphase fluorescence in situ hybridization (FISH) on uncultured amniocytes. Case ReportA 41-year-old woman underwent amniocentesis at 18 weeks of gestation, because of advanced maternal age. Amniocentesis revealed a de novo supernumerary isochromosome 18p in two of 14 colonies of cultured amniocytes. Repeated amniocentesis was performed at 22 weeks of gestation. Interphase FISH analysis on uncultured amniocytes showed four 18p11.32-specific probe (RP11-324G2) signals in 5.7% (3/53 cells) of uncultured amniocytes. A multiplex ligation-dependent probe amplification P095 test kit and array comparative genomic hybridization analysis did not detect genomic imbalance in chromosome 18. Cytogenetic analysis of cultured amniocytes at repeated amniocentesis revealed a karyotype of 47,XY,+i(18)(p10)[3]/46,XY[23]. The pregnancy was carried to 38 weeks of gestation, and a healthy 3120 g male baby was delivered. When examined at 2 months of age, the infant was normal in growth and development, without phenotypic abnormalities. The cord blood had a karyotype of 46,XY. Polymorphic DNA marker analysis excluded uniparental disomy 18. Interphase FISH analysis on uncultured urinary cells showed 9.4% (3/32 cells) mosaicism for tetrasomy 18p. ConclusionThere is cytogenetic discrepancy between amniocytes and cord blood lymphocytes in prenatally detected mosaic tetrasomy 18p. Interphase FISH on uncultured amniocytes has the advantage of rapid confirmation of low-level mosaicism for tetrasomy 18p at amniocentesis.

Virulence and diversity of Peronospora viciae f. sp. pisi in Alberta, Canada

Downy mildew, caused by Peronospora viciae f. sp. pisi, has emerged as a destructive disease of field pea in central Alberta, Canada. The objectives of the current study were to identify the predominant P. viciae pathotypes in Alberta and to assess genetic diversity in regional populations of the pathogen. Three pathotypes (UKP1, UKP2 and UKP11) were identified when nine populations of P. viciae were tested on a host differential set, with pathotype UKP1 being predominant. A collection of 30 P. viciae isolates clustered into three main groups when subjected to random amplified polymorphic DNA (RAPD) marker analysis. Comparison of the internal transcribed spacer (ITS) and 5.8S ribosomal RNA (rRNA) gene sequences revealed a high degree of homology among the isolates, suggesting a common origin. This is the first report on the pathotype composition and genetic diversity of P. viciae in western Canada.

Partial monosomy 21 (q11.2→q21.3) combined with 3p25.3→pter monosomy due to an unbalanced translocation in a patient presenting dysmorphic features and developmental delay

We describe a female patient of 1year and 5months-old, referred for genetic evaluation due to neuropsychomotor delay, hearing impairment and dysmorphic features. The patient presents a partial chromosome 21 monosomy (q11.2→q21.3) in combination with a chromosome 3p terminal monosomy (p25.3→pter) due to an unbalanced de novo translocation. The translocation was confirmed by fluorescence in situ hybridization (FISH) and the breakpoints were mapped with high resolution array. After the combined analyses with these techniques the final karyotype was defined as 45,XX,der(3)t(3;21)(p25.3;q21.3)dn,-21.ish der(3)t(3;21)(RP11-329A2-,RP11-439F4-,RP11-95E11-,CTB-63H24+).arr 3p26.3p25.3(35,333-10,888,738))×1,21q11.2q21.3(13,354,643-27,357,765)×1. Analysis of microsatellite DNA markers pointed to a paternal origin for the chromosome rearrangement. This is the first case described with a partial proximal monosomy 21 combined with a 3p terminal monosomy due to a de novo unbalanced translocation.

Rapid positive confirmation of mosaicism for a small supernumerary marker chromosome as r(8) by interphase fluorescence in situ hybridization, quantitative fluorescent polymerase chain reaction, and array comparative genomic hybridization on uncultured amniocytes in a pregnancy with fetal pyelectasis

ObjectiveThis study aimed at presenting prenatal diagnosis and molecular cytogenetic characterization of a small supernumerary marker chromosome (sSMC) derived from chromosome 8 by fluorescence in situ hybridization (FISH), quantitative fluorescent polymerase chain reaction (QF-PCR), and array comparative genomic hybridization (aCGH) on uncultured amniocytes. Materials, Methods, and ResultsA 32-year-old woman underwent amniocentesis at 19 weeks of gestation because of fetal pyelectasis. Amniocentesis revealed a de novo ring-shaped sSMC in two of 21 colonies of cultured amniocytes. Repeated amniocentesis at 22 weeks of gestation revealed a karyotype of 47,XY,+mar[8]/46,XY[32] in cultured amniocytes. Spectral karyotyping and FISH confirmed that the sSMC was derived from chromosome 8. She underwent a third amniocentesis at 26 weeks of gestation. Oligonucleotide-based aCGH analysis on uncultured amniocytes demonstrated a 43 Mb genomic gain in chromosome 8 encompassing 8p22→q12.1. Polymorphic DNA marker analysis of the uncultured amniocytes revealed a maternal origin of the sSMC and excluded uniparental disomy 8. Interphase FISH analysis showed three D8Z2 signals in 8/40 (20%) of uncultured amniocytes. The cultured amniocytes had a karyotype of 47,XY,+r(8)(p22q12.1)[3]/46,XY[37]. The pregnancy was carried to term, and an apparently normal baby, weighing 3300 g, was delivered with mild hydronephrosis but no other phenotypic abnormalities. The cord blood was found to have a karyotype of 47,XY,+r(8)(p22q12.1)[2]/46,XY[38]. ConclusionPrenatal diagnosis of fetal pyelectasis should alert obstetricians of chromosome aberration. Interphase FISH, QF-PCR, and aCGH analyses on uncultured amniocytes are helpful in rapid positive confirmation of an sSMC detected at amniocentesis.

Pure partial monosomy 3p (3p25.3 → pter): Prenatal diagnosis and array comparative genomic hybridization characterization

ObjectiveThe purpose of this case report is to present prenatal diagnosis and molecular cytogenetic characterization of pure partial monosomy 3p (3p25.3 → pter) by array comparative genomic hybridization (aCGH) and quantitative fluorescent polymerase chain reaction (QF-PCR) on uncultured amniocytes. Case ReportA 35-year-old, gravida 2, para 0, woman underwent amniocentesis at 19 weeks of gestation because of advanced maternal age. Her husband was 37 years of age. She had experienced one intrauterine fetal death. Amniocentesis during this pregnancy revealed a distal deletion of chromosome 3p. The parental karyotypes were normal. Prenatal ultrasonography findings were unremarkable. At 22 weeks of gestation, she underwent repeated amniocentesis, and aCGH investigation using CytoChip Oligo Array on uncultured amniocytes revealed a 9.29-Mb deletion of 3p26.3p25.3 [arr 3p26.3p25.3 (64,096–9,357,258 bp) ×1] encompassing the genes of CHL1, CNTN4, CRBN, LRRN1, ITPR1, and SRGAP3, but not involving the markers D3S1263 and D3S3594. Polymorphic DNA marker analysis on uncultured amniocytes showed a paternal origin of the deletion. Cytogenetic analysis of cultured amniocytes revealed a karyotype of 46,XX,del(3)(p25.3). At 24 weeks of gestation, prenatal ultrasonography findings of the brain, heart, and other internal organs were unremarkable. The pregnancy was subsequently terminated, and an 886-g female fetus was delivered with brachycephaly, hypertelorism, a short and thick nose, micrognathia and low-set ears. ConclusionIn this case, aCGH has characterized a 3p deleted region with haploinsufficiency of the neurodevelopmental genes associated with cognitive deficit and mental retardation but without involvement of the congenital heart disease susceptibility locus, and QF-PCR has determined a paternal origin of the deletion. aCGH and QF-PCR help to delineate the genomic imbalance in prenatally detected de novo chromosome aberration, and the information acquired is useful for genetic counseling.

Development and identification of a wheat-Leymus mollis multiple alien substitution line

Leymus mollis (Trin.) Pilger is resistant to drought, cold, and many fungal and bacterial diseases, and it grows well on barren and salty soils. It has short, strong stems and large spikes with multiple spikelets. It provides an excellent material for wheat (Triticum aestivum L.) improvement. An octoploid Tritileymus line M842-12 was crossed with T. durum L. cv. Trs-372 and among F5 progenies screened a multiple alien substitution line (05DM6) was identified. This line has 2n = 42 chromosomes and forms 21 bivalents in the metaphase I of meiosis. Genomic in situ hybridization analysis of the root tips and pollen mother cells indicated that 05DM6 carried 36 chromosomes from wheat and six chromosomes from L. mollis, and these six, presumed to be from the Ns genome, formed three bivalents. DNA marker analysis showed that relative to wheat, 05DM6 lost chromosomes 1, 5, and 6 from the D-genome but retained chromosomes 2, 3, 4, and 7, implying that the retained L. mollis chromosomes belong to homoeologous groups 1, 5 and 6. Electrophoretic analysis of the storage proteins confirmed that a group 1 homoeologue from L. mollis was present in 05DM6. The 05DM6 substitution line has reduced height relative to its parents and is remarkably resistant to stripe rust. This multiple alien substitution line can be employed as a bridge parent in wheat breeding and chromosome engineering.

Phenotypic and epigenetic changes occurred during the autopolyploidization ofAegilops tauschii

A synthetic autopolyploid was developed from diploid Aegilops tauschii, D genome progenitor of common wheat (Triticum aestivum). The tetraploid Ae. tauschii displayed a markedly larger organ size than the diploid donor. Fluorescence in situ hybridization (FISH) and DNA marker analysis revealed that there is no clear variation at either the chromosomal or DNA level between the diploid and tetraploid plants. We analyzed the variation in cytosine methylation patterns between the diploid and tetraploid plants by methylation-sensitive amplified polymorphism (MSAP) and detected 228 and 232 methylated sites in diploid and tetraploid plants, respectively. Statistical comparison indicated that the tetraploid Ae. tauschii genotype displayed no significant difference in polymorphic methylation level compared to the diploid ones. Twenty-two different genomic fragments displaying different methylation behavior during the ploidy conversions were isolated and sequenced. It demonstrated that alterations in the level of methylation have the most profound effects on coding genes. We demonstrated that there are some genes expressions modified by DNA methylation may be correlated with phenotypic alteration after autotetraploidization.

Production of Donor-Derived Offspring by Allogeneic Transplantation of Spermatogonia in the Yellowtail (Seriola quinqueradiata)1

Although the yellowtail (Seriola quinqueradiata) is the fish most commonly farmed in Japan, breeding of this species has not yet started. This is primarily due to the lack of sufficiently sophisticated methods for manipulating gametogenesis, which makes it difficult to collect gametes from specific dams and sires. If it were possible to produce large numbers of surrogate fish by transplanting germ cells isolated from donor individuals harboring desirable genetic traits, then the probability of acquiring gametes carrying the donor-derived haplotype would increase, and breeding programs involving this species might increase as a result. As a first step, we established a method for the allogeneic transplantation of yellowtail spermatogonia and the production of donor-derived offspring. Donor cells were collected from immature (10-month-old) yellowtail males with testes containing abundant type A spermatogonia, labeled with PKH26 fluorescent dye, and transferred into the peritoneal cavities of 8-day-old larvae. Fluorescence observation at 28 days post-transplantation revealed that PKH26-labeled cells were incorporated into recipients' gonads. To assess whether donor-derived spermatogonia could differentiate into functional gametes in the allogeneic recipient gonads, gametes collected from nine male and four female adult recipients were fertilized with wild-type eggs and milt. Analysis of microsatellite DNA markers confirmed that some of the first filial (F(1)) offspring were derived from donor fish, with the average contribution of donor-derived F(1) offspring being 66% and the maximum reaching 99%. These findings confirmed that our method was effective for transplanting yellowtail spermatogonia into allogeneic larvae to produce donor-derived offspring.

Employment of a new strategy for identification of lemon (Citrus limon L.) cultivars using RAPD markers

DNA markers are useful tools that have potential application in crop cultivar identification and fingerprinting besides others. A novel analysis approach called CID strategy was developed that can realize the utility of DNA marker in the separation of plant individuals much better, more efficiently, practical, and referable, in which a Cultivar-Identification-Diagram (CID) was constructed for fingerprinting. In this study, a total of 47 important lemon cultivars cultivated in China were successfully separated with random amplified polymorphic DNA (RAPD) marker analysis. With the analysis using the CID strategy, they could be clearly separated by the fingerprints of 10 RAPD primers. The utilization of the CID of these 47 lemon cultivars was also verified by the identification of two randomly chosen groups of cultivars among the 47. This identification showed that some advantages in fewer primers were used, and all the cultivars could be separated by each other by the corresponding primers marked in the right position on the CID, and this lemon CID could provide the information to separate any lemon cultivars of these 47 which may be of help to the lemon industry in China. Key words: Lemon cultivar, identification, cultivar-identification-diagram strategy.

Mosaic ring chromosome 21, monosomy 21, and isodicentric ring chromosome 21: Prenatal diagnosis, molecular cytogenetic characterization, and association with 2-Mb deletion of 21q21.1–q21.2 and 5-Mb deletion of 21q22.3

ObjectiveTo present the perinatal findings and molecular cytogenetic characterization of prenatally detected mosaic r(21). Materials, Methods, and ResultsA 29-year-old primigravid woman underwent amniocentesis at 22 weeks’ gestation because of hyperechogenic cardiac foci and intrauterine growth restriction. Amniocentesis revealed a karyotype of 46,XY,r(21)[15]/45,XY,–21[5]. The parental karyotypes were normal. The woman requested repeat amniocentesis. Oligonucleotide-based array comparative genomic hybridization was applied to the uncultured amniocytes, rapidly detecting a 2.09-Mb deletion of 21q21.1–q21.2 (21,495,262–23,580,815bp) and a 5.03-Mb deletion of 21q22.3–q22.3 (41,887,412–46,914,715bp). Cytogenetic analysis revealed a karyotype of 46,XY,r(21)[8]/45,XY,–21[3]/46,XY,idic r(21)[1]. The pregnancy was terminated, and a malformed fetus was delivered with clinodactyly, short big toes, separation between the first and second toes, prominent nasal bridge, downward slanting palpebral fissures, protuberant occiput, prominent forehead, broad anteverted nasal tip, long philtrum, thin upper lip, small mouth, and micrognathia. The placenta had a karyotype of 46,XY,r(21)[83]/45,XY,–21[11]/46,XY,idic r(21)[6], and the cord blood lymphocytes had a karyotype of 46,XY,r(21)[88]/45,XY,–21[9]/46,XY,idic r(21)[3]. Polymorphic DNA marker analysis determined a maternal origin for the deletion. ConclusionAn extra interstitial 21q deletion can be associated with mosaic r(21) in addition to a terminal 21q deletion. aCGH is useful in determining the breakpoints and associated subtle structural abnormalities in cases of prenatally detected ring chromosome in order to facilitate genetic counseling.

Identification of sex in spiny gourd (Momordica dioica) using RAPD marker analysis and its validation in four F1 progenies

SummaryMomordica dioica is an important summer vegetable crop in India and is dioecious. DNA bulks of six male and six female accessions of M. dioica were subjected to random amplified polymorphic DNA (RAPD) marker analysis using 40 primers, of which 22 primers responded for amplification. Of these, ten primers generated reproduction typespecific amplification of DNA fragments at the bulk level. This number was reduced to three RAPD primers when re-tested, individually, with six male and six female accessions. One random decamer primer, OPA-01, produced two unique amplified fragments in all female plants, while two other primers, OPB-04 and OPC-08, generated distinct amplified fragments in both male and female plants. Three amplified fragments (OPB-04430, OPC-081135, and OPC-08578) were unique to all six male accessions, while five amplified fragments (OPA-011560, OPA-01880, OPB-04880, OPB-04800, and OPC-081230) appeared uniquely and uniformly in all six female accessions. Southern hybridisation analysis was performed using five of these sex-specific amplified fragments (OPA-01880, OPB-04880, OPB-04430, OPC-08578, and OPC-081230) as probes with Eco RI and Hind III double-digested DNA of all male and female accessions. Southern hybridisation analysis with the OPC-08578 probe showed a unique hybridisation pattern for all six male accessions, as with the respective RAPD marker, whereas the other probes showed complex hybridisation patterns. Further, inheritance studies also confirmed the presence of a homologous restriction fragment targeted by this probe in F1 progenies, as expected. Hence, OPC-08578 could be used as a marker to ascertain the sex of M. dioica plants at an early stage of development.

Detection and mapping of quantitative trait loci for the contents of the terpenoid indole alkaloids, vindoline and catharanthine, in the leaves of Catharanthus roseus using bulk segregant analysis

SummaryA mapping population consisting of 191 F2:7 recombinant inbred lines (RILs) of Catharanthus roseus was used for segregation analysis of DNA markers, leading to the construction of a genetic linkage map of 174 DNA markers placed on eight linkage groups (LG). These markers were present at an average inter-marker distance of 10.2 cM. Leaves harvested from cultivated C. roseus plants are the main source of vindoline (V) and catharanthine (C), which are chemically condensed during commercial synthesis of the widely-used anti-cancer drugs, vincristine and vinblastine. To detect and map the major genes/quantitative trait loci (QTL) that control the accumulation of V and C in C. roseus leaves, a bulk segregant analysis approach was used. The above mapping population of RILs was phenotyped for their leaf alkaloid profiles and the distributions of V and C contents were used to assemble two pairs of contrasting bulks of RILs, one each for V and C accumulation in extremely low or extremely high amounts. Primers for the 174 already- mapped markers were PCR amplified on the bulks. Among the markers that discriminated between the high-C and low-C bulks, and the high-V and low-V bulks, those that amplified all members of their specific bulk were identified. This led to the detection of two QTLs (V1 and V2) and their co-segregating/linked DNA markers for high-V content, and one QTL (C1) and its linked DNA markers for high-C content. These three QTLs were then mapped. V1 and V2 were observed to lie on LG1, and C1 was on LG6. The QTLs V1 and V2 had large additive effects. These, and C1, appear to be suitable for marker assisted selection.

Acute Regeneration and Chronic Acellular Transformation of Rabbit Cryopreserved Aortic Allografts

An analysis of rabbit cryopreserved aortic allografts excised on postoperative days (POD) 2, 5, 11, 60, 210, 360, and 720, as well as controls that were untransplanted native aortas and cryopreserved aortas, was performed. On POD2, the number of medial smooth muscle cells in the allografts was reduced to approximately 50%. Ki-67 analysis revealed that medial smooth muscle cells in the allografts proliferated from the 2nd day. By the 11th day, their proliferation ceased and the number of medial smooth muscle cells was restored to almost at the same level as in the controls. Polymorphic microsatellite DNA marker analysis disclosed that the restored medial smooth muscle cells were of donor origin. From 7 months through 2 years, the media of cryopreserved aortic allografts were transformed into acellular structures, in which the elastic fibers were preserved. On the other hand, newly accumulated smooth muscle cells were observed in the adventitia just outside of acellular media after 7 months. In some cases, scattered lamellar calcium deposition was observed in the same regions. This study presents a comprehensive documentation of regeneration and acellular transformation in cryopreserved aortic allografts based on short and long-term analysis.